CS266282B1 - A method of radioimmunochemical determination of antigens with barbecue structure - Google Patents

Abstract

The method of radioimmunoassay is suitable for detecting very low concentrations of antigens with a different structure in blood serum, urine, milk, or in extracts from tissue samples or blood. The essence is a unified procedure for radioimmunoassay <·Ί:όΙιο determination of antigens with a different structure, where a goat antibody against rabbit immuno- •j 1 úhu i inu immobilised on modified microcrystalline cellulose is added to a solution of a specific antibody, its antigen labelled with iodine 125 and an unknown sample or standard, always in the same amount and for the same time. This procedure can be used for sensitive radioimmunoassay determination of a free antigen; the separation system is characterised by very good stability in solution, a low value of non-specific binding as well as a low sedimentation rate of the used cleaning agents.

Description

The invention relates to a method for the radioimmunochemical determination of antigens with a sputum structure.

Radio.1 immunoassay (RTA) as a highly sensitive analytical method has found a wide up 1 at since its introduction by Yalovowa and Ihu son ( not in the analysis of a large number of compounds.

The RJA method is based on the ability of an antibody and a specific antigen to form a reversible anti-antigen-antibody complex. A common part of the whole radiochemical assay is the method of combining free and bound antigen. The most commonly used methods include precipitation with polyethylene glycol, ammonium sulfate and other organic compounds, adsorption of free antigen on activated carbon, or use of the so-called a second antibody (animal anti-rabbit immunoglobulin antibody) in combination with polyethylene glycol. The disadvantages of the latter method are usually the relatively complex process of optimizing the separation system for only one type of antigen at a time, the need to add gamma globulin or normal rabbit serum to the reaction mixture and the need for refrigerated centrifuges. The precipitating antibody is not stable! in solution, therefore the manufacturers of RIA kits have to supply it in a lyophilized state and only the customer has to prepare the precipitating agent by mixing several substances.

In addition to the above separation methods, the solid phase method has recently been successfully promoted, i. j. antibodies mobilized on a suitable type of carrier. The most suitable method of immobilization is the covalent attachment of the antibody to a polymeric support (Santos, I., Waerenborgh, F., Patricio, L .: J. of Radional. Nucl. Chem. 102, 143-1986)). In systems where the appropriate type of solid phase is applied, the overall time of the radioimmunochemical assay is generally simplified and shortened.

According to the invention, an animal (preferably goat) anti-rabbit immunoglobulin antibody is immobilized on a support, prepared by crushing microcrystalline cellulose in a ceramic vessel, followed by homogenizing its aqueous suspension in a screened honiogenizer and selecting the finest fraction by flotation; activated by reaction with 1,1'-carbonylimidimidazole. In the radioimmunochemical assay itself, the animal anti-rabbit immunoglobulin antibody, mobilized in this way, is always added in the same volume and concentration to a solution containing the specific antibody, its iodine 125-labeled antigen and the analyzed sample, respectively. standard. Due to the structure of the carrier used and the immobilization method, the method of the invention equilibrates the immunochemical reaction of specific antibody-non-specific antibody in 1 hour at 20 ° C, regardless of whether a low or high molecular weight antigen is determined. The reaction mixture does not need to be stirred or shaken during the incubation, because the sedimentation rate of the support used is sufficiently low. At the end of the one hour incubation, the vehicle with the mobilized animal anti-rabbit immunoglobulin antibody to which the specific antibody-antigen complex is bound is centrifuged, the sediment radioactivity is measured and the content of the antigen in an unknown sample is determined by comparison with values obtained with known antigen concentrations.

The advantage of the determination of antigens with a sputum structure by the method according to the invention is in particular the considerable versatility of the method, the possibility of shortening and simplifying the whole process of radioimmunochemical determination, high degree of reproducibility of results, very good visualization of sediment. Other advantages of the method according to the invention include the possibility of determining antigens with a spruce structure by a uniform procedure using a common precipitating agent, which is ready for use and is characterized by very good stability even in solution - at 4 ° C retains unchanged binding properties for at least 4 months, which is of great technological importance, as this eliminates the lyophilization phase. In the RIA assay itself, it is not necessary to add qammaglobulin, normally rabbit serum or polyethylene glycol, to the reaction mixture, which makes it possible to reduce the number of pipetting steps. A short centrifugation in an uncooled centrifuge is sufficient to separate the free and bound fractions.

CS z'6 6 28 2 Β l

Salting of antigens with a different structure according to the invention, thanks to its universal, also wears in the final! as a result, it significantly saves work capacity, reduces the technical and material demands of optimal use of l..il; the parenteral system as well as the whole radioimmunochemical stand ‘ii. i I · i <Η · · I i (n «11 I ·. 111 y p í í I ·. I, nim i | u ·, · I <Ji <<,, ι I <y

At. k 1 a d I

Pád io i munoana 1 yza ty i o l petroleum hormone (TSH)

The carrier for immunization of the animal antibody against rabbit immunoglobulin is prepared by dry grinding the microcrystalline cellulose in a ceramic vessel. The cellulose is then suspended in sufficient water and homogenized in a glass homogenizer. The 5 to 15 .mu.m particle size fraction obtained by flotation is then separated and dried at a slightly elevated temperature. To 5 parts by weight of the microcrystalline cellulose thus treated, 4 parts by weight of 1,1'-carbonyldiimidazole in 40 parts by volume of acetone are added. Activation takes place for 2 h at 20 ° C. After washing with acetone and 0.05 M phosphate buffer pH 7.5, the activated microcrystalline cellulose is suspended in 40 parts by volume of phosphate buffer pH 7.5 and immediately 5 volumes of animal (goat) anti-rabbit immunoglobulin antibody are added. .1 mobilization takes place in the dark for 24 hours. The iniobilized antibody is then washed with phosphate buffer and diluted 1:10. The precipitating agent thus prepared is also ready for use.

The 200 _f mu spore ifickci anti-TSH was added 200 μΐ standard, respectively. unknown blood serum samples. After preincubation for 3 days at 20 ° C, 1.00 μl of iodine 125-labeled TSH is added to the reaction mixture. The mixture is allowed to stand at 20 ° C for 24 h. This is followed by the addition of 500 μΐ of washing reagent per tube and incubation for 1 hour at 20 ° C. After cross-aging (5 to 10 minutes at 2,000 rpm), the supernatant is removed and the sediment radioactivity is measured. TSH can thus be determined in a concentration range of 0.6 to 40 mlU / l with sufficient sensitivity (0.15 mIU / l) and a low coefficient of variation (up to 5.5%) and non-specific binding (up to 2.7%).

P r i I; 1 and d 2 of the alpha-fetoprotein (AFP) immunoassay

100 (U l specifically) anti-AFP antibody, 100 μΐ pH 7.5 buffer, 100/11 125 1. AFP and 100 (til of standard (samples) are allowed to incubate for 18 hours at 20 ° C. The reaction mixture is then add 500 μΐ of a suspension of immobilized animal antibody against rabbit immunoglobulin, prepared as described in Example 1. After mixing and incubation for one hour at 20 ° C, short centrifugation, aspiration of the supernatant and suction of the gray matter activity.

By this procedure, AFP values in blood serum samples can be determined in the range of 6.25 to 400 ΓΗ / Μ1 with a high sensitivity of the assay (2 IU / ml) and a very low value of non-specific binding (up to 2.5 5).

P r f k 1 a d 3

Radioimmunoassay of tyrogeal lobe (Tg)

To a 200 μΐ standard (unknown serum sample) add 100 μL of specific anti-thyroglobulin antibody. After preincubation for 3 h at 37 ° C, 100 μΐ of 125 g of iodine 125 was added to the reaction mixture and the mixture was allowed to stand overnight at 20 ° C. Then, 500 .mu.l of precipitant, prepared as described in Example 1, was added to each tube. After incubation

266 282 HI

At 20 DEG C. and a short centrifugation center, the supernatant is aspirated and the radioactivity of the sediment is measured.

The। <>;! upem possible with I am </ if values IyiogIohuI i mi in the blood S''r <· in the range 7, 6 to ^r > (l (1 ^ K | 1 <; í · i II i './os 1' on stai io u <-ni. t

P r i k I a d 4

Rád i o i munoa na I y l i ck <'u ta noven i o a Γ I a t ox í nov in milk

Pipette into a test tube after H> 0 μ} of Inu standard ⁽ or an unknown sample), 1 ¹ 5 of a specific antibody against aflatoxin M] and a radioindicator (1 - A FLA Bj). Zincs are zamiosa and incubated overnight at 20 C. ³ Tubes then added to 500 μΐ prccipitačného agent, produced according to Example 1 / the contained? 1 ?. TWEENU 80). The reaction mixture is allowed to incubate at 20 DEG C., the tubes are then centrifuged at 2,000 g for 10 minutes, the supernatant is aspirated and the sediment radioactivity is measured.

This procedure can be used to determine aflatoxins in milk in the range of 0,062 5 to 2,0 (Ug / 1 with a sensitivity of 0,05 A »g / l and a coefficient of variation of up to 5,9 5, with non-specific binding up to 5,5

Example 5

Radiological analysis of ochratoxin A

To 100 μΐ of chloroform extract from a feed, food or tissue sample add} 25

100 μl of radiolabeler solution (1-ochratoxin) and 100 μl of specific] anti-ochrine antibody Í. The mixture is incubated for 1 hour at 45 μl of precipitating agent is then added to each tube and ochratoxin A values in extracts from plant or animal samples in the range of 10 to 320 mg / 100 / can be determined by the same procedure as in the previous examples. [mu] l with an assay sensitivity of 8 mg / 100 [mu] l and a coefficient of variation of up to 5.7%.

Claims (2)

4 H, 2.82 µL of 1 h at 20 ° C and a short center of suction, the supernatant was aspirated and the radioactivity was measured. llvi'11 «n i iisis tip slam-vil 'h'> dn" »y i yio <, i - 0 11 i 1111 in the blood of S''r '· in a range of 6 (10 / in, 1 il 1 1. Os <<a / / / / / / / / / / / / / / / / / 5 / / / / / ád ád ád ád enie enie enie enie enie enie enie enie enie enie enie enie pipette ρο μ 00 μ] of standard milk or unknown sample, specific anti-aflatoxin antibody M, and radioindicator (^ "1 - ΙΕΙ, Λ Bj). Bring the carbon blacks and incubate overnight at 20 ° C. Thereafter, 500 [mu] l of the digestion reagent prepared as described in Example 1 (1 TWEEN 80) was added to the tubes, and the reaction mixture was incubated at 20 [deg.] C. for 1 h, then the flasks were centrifuged at 2000 g for 10 min. the supernatant is aspirated and the radioactivity of the sediment is measured.This method can be used to determine the aflatoxin values in the milk in the range of 0.062 5 to 2.0 µg / L with a sensitivity of 0.05 µg / J and a coefficient of variation of up to 5.9 i, with non-specific binding to 5.5 Example 5 Ochratoxin ® immunoassay 100 To 100 µl of chloroform extract from a feed sample, food or tissue is added 25 100 (1 µl of radioindiate solution (1-ochratoxin) and 100 µl of protiochrotoxin specific antibody Λ). The mixture is incubated for 1 hour at 45. 500 µl of precursor are then added to each flask and the following steps, as in the previous example, can be used to determine the ochratoxin Λ in the extracts of growth or animal samples in the range of 10 to 320 mg / 100 µl with a sensitivity of 8 mg / 100 µl and a coefficient of variation to 5.7%. WE TAKE T Y Y N Λ 1, E Z U 1. A method for radioimmunoassay of antigens having a spiked structure characterized by the fact that both the antibody and the incubator are specific to the antigen. for a suitable period of time with a solution of its antigen marked with iodine 125 and an unknown sample, respectively. Standaid, supersensor and immobilized animal (preferably goat anti-immunoglobulin king anti-homozygous antibodies at the same concentration and volume-free volume for the antigen of interest are added to the assay after the incubation for 1 hour at 20 ° C, and the radioactivity is measured) by comparing the values of the known and concentratory standards with an amount of a given antigen in an unknown sample. 2. A method according to claim 1, wherein the anti-rabbit immunoglobulin animal antibody is immobilized on the surface of the modified microcrystalline cellulose having a particle size of 5 to 15 microns of beacons at a very low settling rate, activated by 1,1-carbonyldiimidazole. Severografia, n. P., MOST Price 2,40 Kcs